Irdye 680

AlloP (3α-hydroxy-5α-pregnan-20-one) was purchased from Steraloids, Inc. (Newport, RI). AlloP was reconstituted in 22.5% (w/v) 2-hydroxypropyl-β-cyclodextrin, purchased from Sigma (St. Louis, MO). Soluble membrane glycoprotein (gp)130 (sgp130) was purchased from R&D Systems (#468-MG) and reconstituted in vehicle (0.1% BSA in saline) as described previously (14 (link)). For intracerebroventricular injections of IL-6 and sgp130, 1.4 μg was dissolved in 100 μl of vehicle to deliver 100 ng per day via a 14 day osmotic pump, at a pump rate of 0.25 μl per hour (Alzet, mini-osmotic pump, model 1002). BSA (0.1%) in saline was used as the vehicle control for both the sgp130 and IL-6 intracerebroventricular injection studies. IL-6 was purchased from Invitrogen and reconstituted in the vehicle (0.1% BSA in saline). Primary antibodies used for immunoblot analysis included NeuN, Calnexin (Millipore), and Cox 4 (Invitrogen), as well as p-STAT3 and STAT3 (Cell Signaling). Secondary antibodies used included goat anti-mouse IRDye 800 and IRDye 680 (LI-COR) and goat anti-rabbit IRDye 800 and IRDye 680 (LI-COR).

White adipose tissue was homogenized in homogenization buffer (150mM NaCl, 10mM Tris-HCl, 2mM EDTA, pH 7,4) and liver tissue in RIPA-buffer (Radioimmunoprecipitation assay buffer 150 mM NaCl, 2mM EDTA, 0,35% Na-deoxycholate, 0,50% Nonidet-40, 0,10% SDS both buffers were supplied with complete protease inhibitor (Sigma-Aldrich, Merk KGaA, Darmstadt, Germany) and phosphoSTOP (Sigma-Aldrich) using a Teflon douncer or a bead beater. Samples were incubated on ice for 30 min and further centrifuged at 10 000xg for 10 min at 4°C and the supernatants were collected.
Total protein concentration was determined using Bradford assay (BioRad). Total proteins were separated using 10 or 12% gradient SDS-polyacrylamide gel (BioRad) under denaturing conditions. Proteins were transferred onto nitrocellulose filters (Nitropure), and blots were probed with antibodies against phosphorylated and total HSL, GAPDH, ASCL1 (Cell Signaling Technology, Inc, Danvers, MA, USA) and FATP2 (abcam, Cambridge, UK), followed by detection by IRDye^TM 800 anti-Goat IgG and IRDye^TM 680 anti-Rabbit IgG antibodies using The Odyssey^® Imaging System (LICOR).

Primary brown adipocytes were lysed in RIPA buffer for western blot analysis. 30 μg of total lysates were separated by SDS-PAGE (12.5% gels), transferred to Odyssey^® nitrocellulose membrane (Millipore), and probed with anti-UCP1 (1:10,000; ab10983, Abcam), and anti-Actin (1:10,000; MAB1501, Millipore). Secondary antibodies conjugated to IRDye^TM 680 or IRDye^TM 800 (Licor Biosciences) were incubated at a dilution of 1:20,000. Fluorescent images were captured by Odyssey infrared imaging system (Licor Biosciences).